Background: Ayurveda, an ancient system of medicine, emphasizes holistic well-being through the balance of doshas and Dhatus. Dhatu Sarata assessment, a critical component in Ayurveda, evaluates tissue strength and quality, guiding personalized treatment strategies. Existing questionnaires for Dhatu Sarata assessment lack standardization and validation, necessitating the development of a reliable tool. Aim: To develop a standard and valid questionnaire for Dhatu Sarata assessment and create user-friendly software for its administration. Objectives: The study aimed to examine available questionnaires, review relevant literature, study classical Ayurvedic texts, consult with subject and statistics experts, and collaborate with software designers to develop and validate a comprehensive Dhatu Sarata assessment tool. Methods: A comprehensive literature review of existing questionnaires and Ayurvedic classics (Brihatrayee, Laghutrayee, Yogratnakar, Bhela Samhita, Harita Samhita, Kashyap Samhita) was conducted. Expert consultations were held to refine the questionnaire. Statistical analysis, including the unpaired t-test, was employed to validate the tool. An Android application was developed and tested for usability and accuracy.